Lateral Intercellular Spaces Research Articles

Development of technical approaches to studing of placental macrophages cultivated In vitro

The theory of Na+ recirculation for isosmotic fluid absorption follows logically from Hertz's convection–diffusion equation applied to the exit of water and solutes from the lateral intercellular space. Experimental evidence is discussed indicating Na+ recirculation based upon the following approaches: (i) An isotope tracer method in small intestine. Simultaneous measurement of water flow and ion transport in toad skin epithelium demonstrating, (ii) occasional hyposmotic absorbates, and (iii) reduced fluid absorption in the presence of serosal bumetanide. (iv) Studies of the metabolic cost of net Na+ absorption demonstrating an efficiency that is lower than the 18 Na+ per O2 consumed given by the stoichiometry of the Na+/K+-pump. Mathematical modeling predicts a significant range of observations such as isosmotic transport, hyposmotic transport, solvent drag, anomalous solvent drag, the residual hydraulic permeability in proximal tubule of AQP1(−/−) mice, the adverse relationship between hydraulic permeability and the concentration difference needed to reverse transepithelial water flow, and in a non-contradictory way the wide range of metabolic efficiencies from above to below 18 Na+/O2. Certain types of observations are poorly or not at all reproduced by the model. It is discussed that such lack of agreement between model and experiment is due to cellular regulations of ion permeabilities that are not incorporated in the modeling. Clarification of these problems requires further experimental studies.

Biosynthesis of basement membrane molecules by salivary adenoid cystic carcinoma cells: an immunofluorescence and confocal microscopic study.

The biosynthesis of basement membrane molecules and fibronectin was studied in vitro in the two different human cell systems (ACC2 and ACC3) established from adenoid cystic carcinomas (ACC) of the salivary gland using immunofluorescence and confocal microscopy. When cells were attached and spread on dishes, fine granular immunofluorescence for type IV collagen, laminin, heparan sulphate proteoglycan, entactin, and fibronectin first appeared diffusely in the cytoplasm, and then changed in aggregation of coarse granules in the perinuclear area. With formation of colonies, these signals were present in the extracellular space, initially in the basal aspect of attached cells and consequently in the lateral intercellular space. After the cells formed a confluent monolayer, extracellular signals started to decrease in inverse proportion to the reappearance of intracellular ones. The results indicate that the parenchymal cells of ACC synthesize these five extracellular matrix molecules, secrete them into the extracellular milieu and remodel the extracellular deposits. It is suggested that the characteristic stromal architecture of ACC, represented by stromal pseudocysts, results from their own secretion of the basement membrane molecules and fibronectin.

Ultrastructural study of the uptake of ferritin by M cells in the follicle-associated epithelium in the small and large intestines of pigs

SUMMARY Uptake of ferritin by M cells in follicle-associated epithelium at various sites in the small and large intestines was examined in 4 healthy 5-week-old pigs by use of electron microscopy. A 2.5% solution of ferritin in saline was injected into ligated loops of the jejunum and ileum containing aggregations of lymphoid follicles (Peyer's patches), as well as into intestinal loops containing lymphoglandular complexes at the ileocecal junction, in the central colonic flexure, and in the rectum. As negative control, saline solution was injected into loops at identical localizations. After an exposure period of 2 hours, uptake of ferritin by M cells, but not by enteroabsorptive cells of the small and large intestines, was observed. Numbers of M cells with ferritin and total M cells were counted and the percentage was calculated. Total number of M cells was highest in lymphoglandular complexes in the rectum and lowest on domes of the ileal Peyer's patch. High numbers of M cells with ferritin were found on domes of the jejunal Peyer's patch, and in lymphoglandular complexes at the ileocecal entrance and in the rectum. Only a few M cells on domes of the ileal Peyer's patch and in lymphoglandular complexes in the central colonic flexure contained ferritin. The percentage of M cells with internalized ferritin was similar on domes of the ileal Peyer's patch, and in lymphoglandular complexes at the ileocecal junction and in the rectum. It was higher on domes of the jejunal Peyer s patches and lower in lymphoglandular complexes of the central colonic flexure. Ferritin was found in the apical tubulovesicular system, multivesicular bodies, and a few vacuoles in the central area of M cells. Ferritin was exocytosed into the lateral intercellular spaces next to M cells. Uptake of ferritin by intraepithelial cells in the follicle-associated epithelium could not be documented, but ferritin was present in vesicles of subepithelial macrophages.

The chloride concentration in the lateral intercellular spaces of MDCK cell monolayers.

We measured the Cl concentration of the lateral intercellular spaces (LIS) of MDCK cell monolayers, grown on glass coverslips, by video fluorescence microscopy. Monolayers were perfused at 37 degrees C either with HEPES-buffered solutions containing 137 mM Cl or bicarbonate/CO2-buffered solutions containing 127 mM Cl. A mixture of two fluorescent dyes conjugated to dextrans (MW 10,000) was microinjected into domes and allowed to diffuse into the nearby LIS. The Cl-sensitive dye, ABQ-dextran, was selected because of its responsiveness at high Cl concentrations; a Cl-insensitive dye, Cl-NERF-dextran, was used as a reference. Both dyes were excited at 325 nm, and ratios of the fluorescence intensity at spectrally distinct emission wavelengths were obtained from two intensified CCD cameras, one for ABQ-dextran the other for Cl-NERF-dextran. LIS Cl concentration was calibrated in situ by treating the monolayer with digitonin or ouabain and varying the perfusate Cl between 0 and 137 mM (HEPES buffer) or between 0 and 127 mM (bicarbonate/CO2 buffer). LIS Cl in HEPES-buffered solutions averaged 176 +/- 19 mM (n = 12), calibrated with digitonin, and 170 +/- 9 mM (n = 12), calibrated with ouabain. LIS Cl in bicarbonate/CO2-buffered solutions averaged 174 +/- 10 mM (n = 7) using the ouabain calibration. The Cl concentration of MDCK cell domes, measured with Cl-sensitive microelectrodes and by microspectrofluorimetry, did not differ significantly. Images of the LIS at 3 focal planes, near the tight junction, midway and basal, failed to reveal any gradients in Cl concentration along the LIS. LIS Cl changed rapidly in response to perfusate Cl with characteristic times of 0.8 +/- 0.1 min (n = 21) for Cl decrease and 0.3 +/- 0.04 min (n = 21) for Cl increase. In conclusion, (i) Cl concentration is higher in the LIS than in the bathing medium, (ii) no gradients of Cl along the depth of LIS are detectable, (iii) junctional Cl permeability is high.

The sodium concentration of the lateral intercellular spaces of MDCK cells: a microspectrofluorimetric study.

MDCK cell monolayers grown on glass coverslips were used to examine the Na+ concentration in individual lateral intercellular spaces (LIS) by video fluorescence microscopy. The LIS was filled with the Na(+)-sensitive fluorescent dye SBFO by incubation of the monolayers for 75-90 min with 250 microM of the membrane impermeant form of the dye. After dye loading, the monolayers were perfused at 37 degrees C with solutions buffered with HEPES or bicarbonate/CO2 containing 142 mM Na+. Ratios of the fluorescence images after sequential excitation with 340 nm and 380 nm light were performed and in situ calibration of LIS Na+ was accomplished after blocking the Na+ pump with 5 x 10(-4) M ouabain. Measurements of Na+ along the basolateral-to-apical axis of the LIS at 1.0 or 1.5 microns intervals did not reveal a Na+ gradient when the perfusate was either HEPES or bicarbonate/CO2 solutions. In bicarbonate solutions, the mean Na+ concentration (mM) was 157.2 +/- 2.3, approximately 15 mM higher than the bath Na+ concentration. In HEPES solutions, however, the Na+ concentration was not different from the bath concentration (142.7 +/- 3.1 mM). The time course of Na+ changes in LIS was investigated by rapidly switching the perfusate from 142 to 80 mM Na+ and measuring the Na+ changes at one focal plane.

Immunocytochemical localization of cathepsin L in the synovial lining cells of the rat temporomandibular joint

Localization of cathepsin L in the synovial lining cells of the normal rat temporomandibular joint was investigated by the avidin-biotin-peroxidase complex method for semithin (1 μm) cryosections and the colloidal gold-labelled IgG method for ultrathin sections of LR gold resin. At the light-microscopic level, type A (macrophage-like) and B (fibroblast-like) cells formed the synovial lining layer. Extensive immunoreactivity for cathepsin L was observed in many granules and vacuoles of type A cells, while in the type B cells, immunoreactivity was found in very few granules. In the sublining layer, macrophages and a few fibroblasts were positive for cathepsin L. By electron microscopy, at the peripheral cytoplasm of the type A cells close to the lateral intercellular spaces and joint cavity, numerous coated vesicles and vacuoles (probably early endosomes) indicating endocytotic function were found. Gold particles indicating cathepsin L were localized in the vesicles (primary lysosomes) in the perinuclear cytoplasm and in the larger amorphous vacuoles (1 μm dia) as phagolysosomes. In type B cells, gold particles were limited to the vesicles only (primary lysosomes). The cathepsin L-positive primary lysosomes were numerous in a few fibroblasts in the sublining layer. These results indicate that type A cells contain a large amount of cathepsin L, and suggest that these cells endocytose surplus substances such as collagen and proteoglycan fragments in normal rat TMJ, effecting their digestion and degradation by the action of this proteolytic cathepsin.

Tight junctions and paracellular permeability in cultured bovine corneal endothelial cells.

Intramembrane specializations of cultured bovine corneal endothelial cells were studied with thin section and freeze-fracture electron microscopy and related to the paracellular permeability and the transendothelial resistance (Rt) of the monolayers. The following intercellular junctions were found: single and discontinuous networks of tight junctions (TJ) which girdle the apico-lateral cell perimeter incompletely, gap junctions, and membrane undulations suggesting intermediate junctions. The macromolecular tracer ruthenium red penetrated into the lateral intercellular space beyond the level of the incomplete belt of TJ. Rt of these monolayers was 20.9 +/- 1.0 omega.cm2. Protamine induced a reversible increase of Rt to 118 +/- 5% of its control value. We conclude that incomplete belts of TJ may be the morphological counterpart of the high paracellular permeability of this monolayer and functionally and morphologically resemble those of their native endothelium. Cultured corneal endothelial cells are an excellent model for studying the influence of incomplete belts of TJ on paracellular permeability of cells.

Acidic pH of the lateral intercellular spaces of MDCK cells cultured on permeable supports

The pH of the lateral intercellular space (LIS) of Madin-Darby canine kidney (MDCK) cell monolayers grown on permeable supports was investigated by microspectrofluorimetry using BCECF (2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein). The permeability of the support was selectively reduced by growing Zn-Al-silicate crystals inside its pores. The diffusion of BCECF across the filter was sufficiently retarded to allow measurements of fluorescence in the LIS. The LIS pH and intracellular pH of the cells surrounding them were determined in HEPES-buffered solutions. When the perfusate pH was 7.4, the LIS pH was more acidic (7.06 +/- 0.02) and equaled the cytoplasmic pH (7.08 +/- 0.05). When perfusate was changed to pH 7.0 or 7.8, the LIS changed linearly by about half the magnitude of the perfusate pH. Intracellular pH followed LIS pH variations between perfusate pH 7.0 and 7.4 but was significantly higher when perfusate pH was 7.8. Tight junctional H+ permeability was undetectably low. The low steady-state pH in the LIS was not altered by inhibitors of acid transport or low temperature. Rapid perturbations of pH in the LIS showed that protons were not immobilized in the LIS. The acidic microenvironment within the LIS may be the result of buffering by the cell surface proteins.

PH, morphology, and diffusion in lateral intercellular spaces of epithelial cell monolayers.

The lateral intercellular spaces (LIS) of reabsorptive epithelia are the site of the proposed local osmotic gradient responsible for transepithelial transport. We developed techniques for loading the LIS of living cultured renal cells (MDCK and LLC-PK1) with the fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF), visualizing LIS geometry, measuring pH, and determining the BCECF diffusion coefficient within the LIS. The LIS pH was remarkably constant and differed substantially from that of the superfusate in both the presence and absence of HCO3 or CO2. The LIS of MDCK cells had a pH of 7.66 +/- 0.04 in bicarbonate-free solutions of pH 7.0, 7.4, or 7.8. In bicarbonate-containing solutions, MDCK LIS pH was acidic to the superfusate by 0.3-0.4 units. In the absence of bicarbonate, the LIS of LLC-PK1 cells was markedly acidic (6.83 +/- 0.05), becoming alkaline by approximately 0.25 units in the presence of bicarbonate. Gradients in pH or dye concentration were not detected within the LIS. The diffusion coefficient of BCECF within the LIS was approximately equal to that seen in free solution.

Localization of Cathepsins B and D in the Synovial Lining Cells of the Normal Rat Temporomandibular Joint by Immuno-light and -electron Microscopy.

The localization of cathepsins B and D in the synovial lining cells and distribution of the lining cells containing these cathepsins in the normal rat temporomandibular joint (TMJ) were examined immunocytochemically in thick (8-10μm) and semithin (1μm) cryosections with the avidin-biotin-peroxidase complex method and in ultrathin sections with the gold-labeled IgG method. In the thick sections, strong immunoreactivity for cathepsins B and D probably in the type A cells (not clear at this level), was observed simultaneously in the superficial layer of the synovial membrane. This reactivity was strong at the following portions of the synovial membrane located adjacent to the blood vessels: 1) at the anterior portion and 2) medial-posterior portion facing the upper joint cavity, and 3) at the anterior portion and 4) lateral portion facing the lower joint cavity. In semithin cryosections, strong immunoreactivity for cathepsins B and D was simultaneously found in the type A cells which have numerous pseudopodia and vacuoles, while weak granular immunoreaction products for both cathepsins were also observed in the type B cells. In control sections, no type A or B cells showed immunoreactivity for these cathepsins. In the ultrathin sections, numerous gold particles indicating cathepsins B and D were detected in the lysosomes and phagolysosomes of the type A cells facing the lateral intercellular spaces and joint cavity. In the phagolysosomes, these cathepsins were characteristically co-localized. On the other hand, in the type B cells, a few gold particles were localized only in the lysosomes.Judging from these findings, it is suggested that type A cells predominantly contain cathepsins B and D, and that these cathepsins may be restricted within the cells (lysosomes) without extracellular release in the normal rat TMJ. It is also suggested that materials such as cell debris in the lateral intercellular spaces between the type A cells or in the joint cavity are endocytosed by phagosomes nearby, and are digested by proteolytic enzymes (cathepsins B and D) in the lysosomes. In addition, it is considered that this function in the type A cells is most conspicuous at the anterior portion of the synovial membrane of the normal rat TMJ.

Tight-junction tightness of Necturus gall bladder epithelium is not regulated by cAMP or intracellular Ca2+

In the preceding publication we have reported that, contrary to the prevailing opinion in the literature, the tight-junction tightness of Necturus gall bladder epithelium is not up-regulated by cAMP-mediated or by Ca(2+)-mediated stimulation. This conclusion was based on our observation that the stimulant-induced increase in transepithelial resistance (Rt) occurred only when the lateral intercellular spaces were allowed to collapse, which suggested that the increase reflected primarily or exclusively the increasing resistance of the lateral spaces (Rlis) rather than the postulated increase in tight-junction resistance (Rj). An alternative explanation could have been that the constancy of Rt after space dilatation reflected an increase Rj that was masked by a concomitant fall in apical and basolateral cell membrane resistances Ra and Rbl. To decide between those possibilities we have performed impedance measurements with transepithelial and intracellular microelectrodes on Necturus gall bladder epithelium. Applying previously developed analysis procedures, the measurements readily showed that elevation of intracellular Ca2+ concentration increased Rlis, but left Rj as well as Ra and Rbl quasi constant. Experiments with forskolin, theophylline or isobutylxanthine, on the other hand, were less clear. These stimulants activated an apical Cl- conductance, which drastically reduced Ra and apparently caused low-frequency polarization effects that could not be accounted for by the classical epithelial equivalent circuit. After elimination of the polarization phenomena by uni- or bilateral substitution of Cl- by isethionate or sulphate, however, we were able to demonstrate that Rj remains constant under cAMP-mediated stimulation irrespective of whether the lateral spaces are kept open or are allowed to collapse. We conclude that the tight-junction resistance of Necturus gall bladder epithelium is not controlled by intracellular Ca2+ or by cAMP-mediated stimulation.

Long-term effects of tumor necrosis factor on LLC-PK1 transepithelial resistance.

Renal epithelial LLC-PK1 cell sheets incubated with tumor necrosis factor (TNF) undergo an acute, spontaneous, and rapidly reversible decrease in transepithelial resistance (TER). (Mullin et al., 1992). However, 24 to 72 h following TNF exposure, TER across the cell sheet increases 2-fold. This later effect of TNF is also reversible, albeit slowly. The TER of TNF-treated cell sheets then declines toward initial levels between 72 and 144 h following exposure to the cytokine. Whereas the long-term increase in TER following TNF exposure is not associated with a decreased transepithelial 14C-mannitol flux (size selectivity), the charge (anionic) selectivity of the LLC-PK1 tight junction is decreased. Basal-lateral (ouabain and bumetanide-insensitive) Rb+ and apical Na+-dependent alpha-methylglucoside (AMG) uptake into the cell are both reduced in cultures exposed to TNF 24 h earlier. Correspondingly, this long-term effect on TER is accompanied by a 30% decrease in short circuit current (iscc). Along with an observed increase in basal-lateral methylamino-isobutyric acid (MeAIB) influx into the cells, an increased incorporation of [3H]-thymidine into DNA indicates increased cell cycling after exposure to TNF. While the increase in cell cycling is not sustained for the duration of the elevation in TER, it does appear to initiate a sequence of events that lead to the sustained increase in TER. A decrease in the lateral intercellular space, observed between these epithelial cells after long-term TNF exposure, may be a mechanism for the elevated TER following from the mitogenesis and/or transport changes. This overall long-term tightening of an epithelium in response to TNF may function, in part, as a compensatory action of the epithelium to reestablish its effectiveness as a physiological barrier, following the acute effect of TNF.

Ultrastructure and barrier properties of the endolymphatic duct in the guinea pig.

The ultrastructure and barrier properties of the endolymphatic duct (ED) were examined by light and electron microscopy. ED epithelial cells were classified into two types: type I and type II duct cells. The type I duct cells were cuboidal or low columnar and were characterized by a convex apical surface and a few basal processes. The type II duct cells were squamous and were characterized by a flat apical and basal membrane, many small vesicles and a number of small pits along the basal membranes. After electrophoretic horseradish peroxidase (HRP) injection into the ED lumen, no HRP uptake into ED epithelial cells of either type was observed. There was no reaction product either in the lateral intercellular spaces beyond the tight junctions between ED epithelial cells or in ED subepithelial tissues. The ED epithelial cells were considered to play no active role in apical macromolecular absorption and to be impermeable to intraluminal macromolecules.

Distribution of anionic sites during increasing tight junctional permeability in the rat submandibular gland.

We sought to determine the effect of substance P salivary stimulation on both electrical charges and cellular permeability in the tight junctions of rat submandibular gland cells. Microperoxidase (1,900 daltons) was used as a tracer. It was administered by close-arterial infusion via the glandular arteries, and secretory routes of acinar cells in the gland were determined cytochemically. In the resting gland, microperoxidase reaction product filled the lateral intercellular spaces up to the tight junctions, but did not penetrate them. In the substance P-stimulated gland, microperoxidase reaction product was present within tight junctions and the lumen. Both distribution and mobility of anionic sites on the surface of the submandibular gland cells were studied utilizing multivalent ligand, ruthenium red and cationized ferritin as probes. In the resting gland, ruthenium red deposits were located uniformly in all areas of the basal membrane and intercellular spaces except for the tight junctional region of acinar and ductal cells. In substance P-stimulated gland, ruthenium red deposits were present in the tight junctional region and, to a lesser extent, in the intercellular spaces. Electrical charges of the tight junctions area of the lateral plasma membrane were studied using intraductal injection of cationized ferritin. In the resting gland, cationized ferritin probe was present in the intercellular spaces and was bound weakly in the tight junctional region. In the substance P-stimulated gland, cationized ferritin was firmly adherent to the tight junctional region.

Ultrastructure of the endolymphatic duct in the rat. Fixation and preservation.

Ten rats were vascular-perfused at subphysiologic as well as physiologic pressures, 80 mmHg and 120 mmHg, respectively, employing a pressure feed-back controlled peristaltic pump and an isotonic perfusate/fixative with colloids (2% Dextran) and a hypertonic perfusate/fixative without colloids, 300 and 530 mOsm, respectively. In both experiments the endolymphatic duct and sac were isolated by microdissection after primary fixation. When comparing micrographs from the two experiments we observed that rats perfused at low pressure in isotonic fixative with colloids added had non-dilated lateral intercellular spaces and a subepithelial ground substance loaded with uniformly arranged microfibrils, not previously discovered. In rats perfused at high pressure in hypertonic fixative with no colloids added, we observed an edematous state in the subepithelial space between the solid bony aqueduct and the endolymphatic duct, with a concomitant derangement of previously well organized microfibrils. Furthermore, the epithelium was quite clearly displaced from the capillaries, the intercellular spaces were widely dilated and the endolymphatic duct was compressed into its lumen. Methods of fixation and current theories of endolymph resorption by the endolymphatic duct are discussed.

Acute effects of ethane dimethane sulfonate (EDS) on the structure of the cauda epididymidis in the rat: selective destruction of clear cells in the proximal cauda region.

Of eight groups of five adult male Sprague-Dawley rats, three groups received an intraperitoneal dose of EDS (75 mg kg-1) in DMSO (dimethylsulfoxide in water; 1:3) and three control groups received DMSO only. One EDS and one control group were killed 1, 2 and 3 days after treatment. One EDS+T group, given a 5-cm implant of T sufficient to maintain normal serum T concentrations, and one untreated control group were killed on Day 3. Epididymides were fixed by vascular perfusion and embedded in Epon/Araldite for light and electron microscopic studies. Epididymal duct diameters and epithelial heights were measured from 1-micron sections and structural changes were assessed from thin sections using a Jeol 100B electron microscope. Morphological studies showed a reduction in duct diameter and an associated increase in epithelial height in the proximal cauda epididymidis of the experimental groups. In the EDS+T implant group, epithelial heights were significantly greater than in controls but duct diameters remained unchanged. The most obvious structural change in the proximal cauda epididymidis was the selective destruction of clear cells in the epithelium. Initially, vacuoles were observed in the lateral intercellular spaces of the epithelium; large autophagic vacuoles then appeared in the clear cells, which had degenerated and disappeared from the epithelium by Day 3. Progressive infiltration of leucocytes into the intertubular interstitium, the epithelium and lumen of the proximal cauda was also observed. Loss of clear cells in the proximal cauda epididymidis was also seen in the EDS+T group, suggesting that clear-cell degeneration was not associated with reduced concentrations of circulating androgen. In all EDS-treated groups, however, clear cells and duct profiles in the distal cauda epididymidis remained unaffected. The reason for their protection from the effects of EDS has not yet been determined. These results suggest that, in addition to other specific lesions described in previous studies, EDS also has a direct effect on the rat epididymis that appears to be specifically targeted to the clear cells in the proximal caudal region.

The tight junction: Structure and function

The tight junction (TJ) is a specialized structure that generally circumscribes the most apical region of the lateral plasma membranes of polarized epithelia and endothelia. It apparently consists of two sets of strands, one for each adjacent cell, which seal the lateral intercellular spaces, thus restricting the passage of substances through the paracellular pathway. It also forms a boundary between the apical and basolateral plasma membrane domains. Although polarization of membrane components may proceed independently of the TJ, they apparently segregate certain proteins and lipids into their respective domains, thus preventing their intermixing. The molecular nature of the sealing element is still unknown, but proteins such as ZO-1 and cingulin have recently been isolated and detected in association with the cytoplasmic side of the TJ. A variety of data now suggest that TJ are highly dynamic structures, sensitive to envronmental and cellular factors. They are able to control the paracellular pathway depending on physiological requirements, presumably through regulatory mechanisms and interactions with the cytoskeletal microfilament system. Their assembly and sealing may depend on extracellular Ca 2+, cell-adhesion mechanisms, recognition and cascades of cellular events yet to be determined. Determination in the near future of the molecular composition of the sealing element in TJ and its control mechanism will undoubtedly contribute to the basic understanding of these important membrane specializations in health and disease.

Driving forces and pathways for H+ and K+ transport in insect midgut goblet cells.

In the midgut of larval lepidopteran insects, goblet cells are believed to secrete K+; the proposed mechanism involves an electrogenic K+/nH+ (n > 1) antiporter coupled to primary active transport of H+ by a vacuolar-type ATPase. Goblet cells have a prominent apical cavity isolated from the gut lumen by a valve-like structure. Using H(+)- and K(+)-selective microelectrodes, we showed that electrochemical gradients of H+ and K+ across the apical membrane and valve are consistent with active secretion of both ions into the cavity and that the transapical H+ electrochemical gradient, but not the transapical pH gradient, is competent to drive K+ secretion by a K+/nH+ antiporter. We used 10 mmol l-1 tetramethylammonium ion (TMA+) as a marker for the ability of small cations to pass from the gut lumen through the valve to the goblet cavity, exploiting the high TMA+ sensitivity of 'K(+)-sensitive' microelectrodes. These studies showed that more than half of the cavities were inaccessible to TMA+. For those cavities that were accessible to TMA+, both entry and exit rates were too slow to be consistent with direct entry through the valves. One or more mixing compartments appear to lie between the lumen bathing solution and the goblet cavity. The lateral intercellular spaces and goblet cell cytoplasm are the most likely compartments. The results are not consistent with free diffusion of ions in a macroscopic valve passage; mechanisms that would allow K+ secreted into the goblet cavity to exit to the gut lumen, while preventing H+ from exiting, remain unclear.

Deficiency of red cell sodium-potassium pump activity in bipolar affective disorder

The rat proximal tubule epithelium is represented as well-stirred, compliant cellular and paracellular compartments bounded by mucosal and serosal bathing solutions. With a uniform pCO2 throughout the epithelium, the model variables include the concentrations of Na, K, Cl, HCO3, H2PO4, HPO4, and H, as well as hydrostatic pressure and electrical potential. Except for a metabolically driven Na-K exchanger at the basolateral cell membrane, all membrane transport within the epithelium is passive and is represented by the linear equations of nonequilibrium thermodynamics. In particular, this includes the cotransport of Na-Cl and Na-H2PO4 and countertransport of Na-H at the apical cell membrane. Experimental constraints on the choice of ionic conductivities are satisfied by allowing K-Cl cotransport at the basolateral membrane. The model equations include those for mass balance of the nonreacting species, as well as chemical equilibrium for the acidification reactions. Time-dependent terms are retained to permit the study of transient phenomena. In the steady state the energy dissipation is computed and verified equal to the sum of input from the Na-K exchanger plus the Gibbs free energy of mass addition to the system. The parameter dependence of coupled water transport is studied and shown to be consistent with the predictions of previous analytical models of the lateral intercellular space. Water transport in the presence of an end-proximal (HCO3-depleted) luminal solution is investigated. Here the lower permeability and higher reflection coefficient of HCO3 enhance net sodium and water transport. Due to enhanced flux across the tight junction, this process may permit proximal tubule Na transport to proceed with diminished energy dissipation.

Video microscopic measurements of diffusion and flow in cultured kidney cells

New, optical microscopic techniques were developed to study the composition, fluid flow patterns and restrictions to diffusion in the fluid filled spaces surrounding renal epithelial cells. The prevailing theoretical model for the mechanism of isosmotic fluid transport by epithelia, the standing osmotic gradient hypothesis, predicts gradients in ion composition as well as significant restrictions to the diffusion of small solutes within the lateral intercellular spaces separating epithelial cells. Key questions about the validity of the model arise from uncertainties about the values for the hydraulic water permeability of the cell membranes and the lack of data about the composition of the fluid filling the lateral intercellular spaces. This presentation will describe measurements of the local composition, geometry and diffusion coefficients within lateral intercellular spaces of cultured renal epithelia and in the lumen of isolated, perfused renal tubules.